16alpha-methyl-1, 4-pregnadiene-3, 20-diones



United States Patent 3,354,186 Mat-METHYL-1,4-PREGNADIENE-3,20-DIONES Gerhard Rasp and Klaus Kieslich, Berlin-Charlotteuburg,

Erich Olivar, Berlin-Friedman, Rudolf Miiller, Berlin- Lankwitz, and Brigitte Wagner, Berlin-Haleusee, Germany, assignors to Schering A.G., Berlin, Germany No Drawing. Original application May 2, 1961, Ser. No. 107,029, now Patent No. 3,102,080, dated Aug. 27, 1963. Divided and this application Oct. 31, 1961, Ser. No. 148,810 1 Claims priority, application Germany,

May 20, 1960, Sch 27,920 Claims. (Cl. 260-397.45)

This application is a division of our copen-ding application Ser. No. 107,029, filed May 2, 1961, now Patent No. 3,102,080 entitled Method of Producing 1,4-diene- I i-ketosteroids.

The present invention relates to a method of producing 1,4-diene-3-ketosteroids, and more particularly to a new method which results in the production of such 1,4-diene-3-ketosteroids in high yield and also starting from compounds which could not previously be used as starting materials for the production of such final products, as well as to the production of new compounds.

The various perhydrocyclopentenophenanthrenes which simultaneously have double bonds in the l-position and in the 4-position, for example prednisolone, prednisone, triamcinolone, A -androstadiene-3J7-dione, and many others, possess valuable properties which make these compounds of great interest as therapeutic products and also as intermediate products for the production of therapeutic steroid products.

Methods of producing these compounds comprise not only chemical methods but also microbiological methods, the latter having the advantage also that they are applicable to compounds which are sensitive against chemical reagents, and in addition often gives rise to considerably improved yields. Not only bacteria but also fungi have been used for the introduction ofa A -double bond.

The species belonging to the family Bacillaceae have the common property of spore formation. With respect to the biochemical activity not only do the various species difler from each other, but also the strain of each species vary considerably, and in contrast to the fungi it is not possible by placing a bacteria in its place in the system to predict what its biochemical activity would be since often the origin of the individual strain of a species is of considerable importance with respect to its biochemical activity.

Thus, for example, a great number of hitherto available strains of Bacillus subtilis (Naturwissenschaften, 43, page 39) do not possess the property which they would be expected to possess according to the publication, namely of dehydrogenating a steroid hormone in 1,2- position.

In the case of the species Bacillus sphaericus, which has been used for a long time for the purpose of 1,2-dehydrogenation in the steroid series, it has been shown that besides very slow reaction speed that the success of the epiandrosterone into A -androstadiene-3,17-dione. However, with these microorganisms only extremely moderate cause of the slow reaction speed.

yields can be obtained and these require a long timebe- Ice The use of Fusarium solani or Strcptomyces lavendulae is also disadvantageous for the conversion of dehydroepiandrosterone to A -androstadiene-3,l7-dione because in addition to the desired A -androstadiene-3,17- dione also large amounts of testololactone and 1,2-dehydrotestololactone are formed.

It is accordingly a primary object of the present invention to provide for the production of A steroids in high yields and with improved reaction speed.

It is another object of the present invention to provide a microbiological method of producing A -steroids which can start from various different starting compounds which could not be used as starting compounds prior to this method.

It is yet another object of the present invention to provide for the use of a new mutant of a microorganism which is particularly advantageous in the production of A -steroids.

A still further object of the present invention provides for the method of producing such microorganism mutant for this purpose. As yet another object the present invention provides for the production of new compounds which could not be produced prior to the method of the present invention. Other objects and advantages of the present invention will be apparent from a further reading of the specification and of the appended claims.

With the above and other objects in view, the present invention mainly comprises the discovery that a specially produced mutant of the Bacillus lentus has extremely good properties in the production of A -steroids starting from various different starting compounds.

It has been discovered that it is possible to produce from per se inactive strains of the family Bacillaceae, preferably of the genus Bacillus, specially of the species Bacillus lentus by choice artificially produced mutants of the species which exhibit a remarkably improved biochemical activity.

It has thus been discovered that by ultra violet irradiation of a strain of the species Bacillus lentus which with respect to 1,2-dehyd'rogenase activity has at most only slight activity, which is isolated from a compost, to obtain by systematic selection a mutant (MB 284) which not only has the desired activity of dehydrogenation in the 1,2-position in singular manner, but in addition is capable of converting a 3-hydroxy-A -group or a 3-acyloxy- A -group into the 3-keto-A -group. This obviously means a great extension of the groups of suitable starting materials for the production of A -3-ketosteroids.

The starting compounds for the method of the present invention may be designated as any steroid the A ring of which has the following structure:

A X s wherein either Y or Z designates a carbon-carbon double bond to the S-carbon atom, and wherein X is selected from the group consisting of t ell d a 0 H0 an AcylO By treatment of such starting compound with Bacillus lentus MB 284 in accordance with the method of the present invention the resulting compound will be the corresponding steroid'wherein the A ring has the following structure:

A culture of Bacillus lentus MB 284 was deposited at the American Type Culture Collection in Washington, D.C., under No. 13,805.

The surprising property of Bacillus lentus MB 284 does not correspond with any other known strain of the family Bacillaceae, and it also could not be predicted that by artificial mutation of a per se inactive strain that it would be possible to obtain a mutant with improved biochemical properties.

It has further been found that upon fermentation with the mutant MB 284 not only are surprisingly higher yields obtained, but also undesired conversion of the D-ring to a lactone ring does not occur and as a result the speed of the reaction is increased by about times to about 7 times.

Within the above mentioned broadened group of suitable starting materials are included perhydrocyclopentenophenanthrene compounds which are saturated in the 1,2-position which contain at least one hydrogen atom on each of the carbon atoms in 1 and 2 position, preferably however containing 2 hydrogen atoms on each of the 1- and 2-ca-rbon atoms, as well as compounds which contain on one of the two carbon atoms one hydrogen atom and on the other two hydrogen atoms, which compounds also contain an oxygen-function on the 3-carbon atom, preferably a keto group, a hydroxy group or an acyloxy group, most preferably a lower acyloxy group such as the acetyloxy group, in combination with a double bond between the 4 and 5 carbon atoms or between the 5 and 6 carbon atoms. It should be noted that these conditions do not exclude the possibility that at another portion of the steroid molecule there may be present other double bonds, and/or on the IO-carbon atom a methyl group, a free or functionally changed hydroxyl group or only a hydrogen atom, and that on still other carbon atoms such as the carbon atoms in the 6-, 9-, 11-, 16-, 17-, 20- or 21-position of the steroid framework there may be present the usual substituents such as keto groups, hydroxyl groups, methyl groups, epoxy groups, or halogens, particularly fluorine.

The further biochemical properties of the Bacillus lentus mutant MB 284 of the present invention are set forth in the following table in comparison to the species Bacillus lentus (according to Bergeys, Manual of Determinative Bacteriology, 1957):

TABLE 1 Bacillus lentus Bacillus lentus 5% glucose-[05% potassium nitrate agar slant.

1.5% soya bean meal agar slant.

Nutrient bouillon.

Milk agar ruled plate.

0. 5% ammonium nitrate. Nitrate reduction.

High gelatin 1ayer Gelatin plate Sodium nutrient,

No growth Slight growth Good growth, uniform turbidity, granular sediment.

Casein is not hy- Is not formed, no gas formation.

No liquification 4% good, 5% no growth.

Gas formation None...-.

Very slight growth.

Slight growth.

Good growth, uniform turbidity.

Casein not hydrolyzcd. Unchanged. Slight growth. No hydrolysis. Is not formed. No growth.

slight growth. None.

TABLE 1Contlnued Bacillus lentus Bacillus lentus Meat extract +05% As nitrogen sourc No acid formation occurs.

The following abbreviations are used in the table:

Ar.=Arabinose Xy.=Xylose Gl.=Glucose La.=Lactose Ma.-=Mannose So.=Sorbose Sc.=Saccharose +-=Acid formation O=No acid formation =Slight acid formation The following examples are given to further illustrate the present invention. The scope of the examples are not, however, meant to be limited to the specific details of the examples.

EXAMPLES General method A 50-liter capacity stainless steel fermenter is charged with 30 liters of the nutrient solutions mentioned in the following table; the nutrient solution is sterilized by heating for one half hour at C., and after cooling inoculated with a bacteria suspension which is obtained by rinsing a bouillon agar surface of 64 cm. with 7 cc. of physiological saline solution.

After 2 days of culturing at 25 C. under stirring (220 revolutions per minute) and airing (1650 liters per hour) 1.8 liters or the resulting culture are removed under sterile conditions and transferred into a fermenter with 28.2 liters of the same medium. At the same time there is added a solution of 7.5 g. of the steroid mentioned in the table in 200 cc. of ethanol and fermented under the same conditions. The fermentation times depending upon the particular steroid are also set forth in the table, and it will be seen that these times vary.

The course of the fermentation is followed by removing samples which are extracted with methylisobutyl-ketone. The extracts are analyzed by paper chromatography, preferably using a system of dioxane-l-toluene/ propylene glycol and heptane/ propylene glycol.

At the end of the fermentation time the culture broth is extracted three times, each time with 10 liters of methylisobutyl ketone. The purified extracts are concentrated in a circulating evaporator under vacuum and then under vacuum in a nitrogen atmosphere evaporated to dryness. The residue is subjected to chromatography on silica gel (10% addition of water). The utilized eluation agent as well as the solvent for the recrystallization of the isolated substance are set forth in the table which follows.

The nutrient solution Md. 7 has the following composition:

The fermentation temperature may in general vary between about 20 and 30 C., and is most preferably maintained at 25 C. The preferred concentration range is between 100 and 200 mg./l., and is most preferably about 250 mg. per liter.

Although the above set forth fermentations were all carried out with cultures, as indicated previously, the reaction may be carried out by the use of enzymes which can be obtained in per se known manner by mechanically destroying the cells or by destroying them by means of ultra-sonics and subsequently fractionally precipitating (for example by means of ammonium sulfate, protamine sulfiate, etc.) or by fractional absorption (for example, with calcium phosphate gel). This is preferably carried out at temperatures between and 10 C.

' Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential char- :acteristics of the generic or specific aspects of this invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. 16u-methyl-1,4-pregnadiene 116,170: diol 3,20- dione.

2. l6a-methyl-1,4pregnadiene-l1fi,2l-di0l3,20-dione. 3. 16a-methyl-1,4-pregnadiene-17a-0L3,l1,20-trione.

4. l6a-methyl 1,4-pregnadiene-17a-ol-3,11,20-trione- Not-acetate.

5. 16u-methyl-1,4,-pregnadiene 116,21 diol 3,20 dione-Zl-acetate.

References Cited UNITED STATES PATENTS 2,887,499 5/1959 Carvajal 260397.45 3,023,206 2/1962 Burn et al. 260-23955 3,161,663 12/1964 Joly et1al 260-397.45

' FOREIGN PATENTS 850,368 10/1960 GreatBritain. 329,570 6/1958 Switzerland.

OTHER REFERENCES Oliveto, I.A.C.S., V01. 80 (1958) p. 4428 relied on.

ELBERT L. ROBERTS, Primary Examiner.

M. LIEBMAN, IRVING MARCUS, LEWIS GOTTS,

Examiners. 

1. 16A-METHYL-1,4-PREGNADIENE-11B,17A-DIOL-3,20DIONE. 